IgG SUBTYPING ASSAY FOR IDENTIFYING TRANSPLANTABLE TISSUE SAMPLES

ABSTRACT

The disclosure relates to methods for assessing the suitability of a tissue obtained from a vertebrate (e.g., human) donor for grafting to a recipient, such as another vertebrate of the same species. The method involves contacting leukocytes obtained from the donor with serum from the recipient. Binding between recipient antibodies and the leukocytes is assessed, specifically focusing on the subtype(s) of IgG antibodies which bind with donor leukocytes. Detected binding between recipient antibodies of subtypes IgG1 and IgG3 indicates that the donor tissue is not suitable for grafting to the recipient. Detected binding between recipient antibodies of subtypes IgG2 and/or IgG4 indicates that the donor tissue can suitably be grafted to the recipient.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is entitled to priority to U.S. Provisional PatentApplication No. 62/262,636 filed Dec. 3, 2015 and is a continuation ofU.S. patent application Ser. No. 15/369,097 filed Dec. 5, 2016.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND OF THE DISCLOSURE

The invention relates generally to the field of organ and tissuetransplantation, and more specifically to procedures for matching donortissues and recipients whose bodies will not reject the tissue iftransplanted into the recipient.

Transplantation of organs and tissues from the body of a donor into thebody of a recipient has become a relatively common procedure. Owing tofundamental functions of the immune system of vertebrates (i.e., toalert the body to “non-self” materials and to assist in elimination ofsuch non-self materials), tissues cannot be simply transplanted from thebody of one individual of a given species into the body of any otherindividual of the same species without the possibility of immunecomplications. If the transplanted tissue is not compatible with theimmune system of the recipient, the recipient's immune system can mounta response against the transplanted tissue whereby the transplantedtissue is destroyed. In order to avoid such transplant rejection, twobasic strategies can be employed. First, one or more immune functions ofthe recipient can be inhibited, so as to lessen or eliminate the immuneresponse mounted against the transplant. Second, the transplant andrecipient can be screened and matched, to lessen the likelihood and/orseverity of a recipient immune response against the transplant. Thesubject matter described herein is directed primarily toward this secondstrategy, although the two strategies are not mutually exclusive—bothcan be employed in the same transplantation.

It has long been known that cells in vertebrate tissues express antigenson their surface, and that these antigens can vary significantly amongindividuals of the same species. For example, human cells displayproteins designated human leukocyte antigens (HLAs) on their surfaces.For any individual human, the HLAs displayed by that individual's cellsare recognized as “self” by the individual's immune system, and theindividual's immune system does not (normally) mount an immune responseagainst his or her own cells. However, the HLAs displayed by differentindividual humans can vary significantly enough that HLAs not normallyexpressed by an individual's cells, such as HLAs of transplanted tissue,can be recognized as “non-self” by an individual's immune system,leading to initiation of an immune response directed against thetransplanted tissue. If the immune response initiated against atransplanted tissue is sufficiently severe to induce death of most orall transplanted cells or destruction of sufficient transplantedextracellular material, the transplanted tissue can fail to exhibitdesirable functions or properties that were the reason for thetransplant. That is, the goals which led to the transplantation can failto be achieved if the recipient immune response mounted against thetransplanted tissue is sufficiently severe.

One type of immune response that a recipient's body can mount against atransplanted tissue depends on binding of antibodies produced by therecipient's body to antigens (e.g., HLAs) that are displayed on surfacesof the transplanted tissue. Specific binding between antibodies andtheir corresponding antigens can catalyze a reaction (“complementfixation”) that leads to induction of a potent cytotoxic immune responsedirected against the transplanted tissue. Analyzing the ability ofrecipient immune system components to detect donor tissue as “non-self”and mount a cytotoxic response and selecting donor-recipient pairs toavoid such reactions is referred to generally as “crossmatching” and isdiscussed extensively in the literature (see, e.g., Mulley et al., 2011,Nephrology 16:125-133).

One known crossmatching technique involves contacting lymphocytesobtained from the donor of a potential transplant tissue with bloodserum obtained from a proposed recipient of the transplant tissue. Serumincludes antibodies which circulate in the blood of the recipient. Ifthe recipient's serum includes antibodies which bind specifically withantigens which appear on donor lymphocytes, such binding can bedetected. In one common detection technique known as “flowcrossmatching,” donor lymphocytes are contacted with recipient serum fora period of time, after which non-bound antibodies are removed byseparating the lymphocytes and serum and rinsing the lymphocytes with anexcess reagent. The lymphoctyes are then contacted with a labeledreagent capable of detecting recipient antibodies (e.g., afluoresceinated antibody which binds to human antibodies, optionally ofa particular type, such as IgM or IgG, or subtype, such as IgG1, IgG2,IgG3, or IgG4) and thereafter rinsing non-bound labeled reagent from thelymphocytes. Binding of recipient antibodies with donor lymphocytes isdetected by suspending the lymphocytes in a fluid and passing themthrough a flow cytometer capable of detecting the label of the labeledreagent. Detection of the label together with a lymphocyte by the flowcytometer (e.g., detection of fluorescence corresponding to fluorescein)indicates that one or more recipient antibodies recognized by thelabeled reagent is bound to the lymphocyte. Such bound recipientantibodies are commonly designated “anti-donor antibodies” (ADAbs). Ifmultiple flow crossmatching reactions are performed, each using alabeled reagent having specificity for a different recipient antibodytype and/or subtype, a profile of the type(s) and subtypes(s) of ADAbsthat are present in an potential recipient's serum can be developed.

It has been observed that ADAb types and subtypes can influence thelikelihood that a transplanted tissue will be rejected by a recipient.Mulley et al., for example, recognized that IgG4 does not activatecomplement and that potential recipients whose ADAb detected using donorleukocytes are solely of the IgG4 subtype may be less likely to activatecomplement in vivo (i.e., indicating a potentially suitabledonor-recipient match). Comparable observations were reported byCicciarelli et al. (U.S. patent application publication no.2010/0261203). Gao et al. (2014, Am. J. Transplant. 14(7):1581-1591)recognized that IgG antibodies of subtypes IgG1 and IgG3 which reactwith apoptotic cells are more likely to lead to late rejection oftransplanted kidneys, presumed by those authors to be attributable tothe complement-fixing ability of IgG1 and IgG3 subtypes. However,previous workers examined binding between antigen-coated beads andantibodies in recipient serum, which assays can be laborious and notnecessarily indicative of interactions between recipient antibodies anddonor cells.

Improved crossmatching assays capable of quickly and accuratelydetermining the suitability of a potential tissue transplant forimplantation in (or on) an individual recipient would be desirable. Thisdisclosure describes such assays.

BRIEF SUMMARY OF THE DISCLOSURE

The invention relates to methods of assessing compatibility of a bodytissue of a potential donor for grafting with a recipient. The methodsinvolve contacting leukocytes obtained from the potential donor withantibodies obtained from the recipient. Binding between the potentialdonor leukocytes and recipient antibodies is assessed for at least IgGsubtypes IgG1 and IgG3. The body tissue is assessed as compatible forgrafting with the recipient if substantially no binding between thedonor leukocytes and recipient antibodies of either of IgG subtypes IgG1and IgG3 is detected. The invention also involves tissue graftingprocedures which include such assessment methods.

In the assessment methods, binding between the donor leukocytes andrecipient antibodies is preferably assessed for each of at least IgGsubtypes IgG1, IgG2, IgG3, and IgG4, preferably usingdifferentially-labeled antibodies that distinguishably recognize thesubtypes, and preferably in a single assay mixture. If desired, theassessment methods can also include contacting the donor leukocytes withdifferentially-labeled antibodies that distinguishably recognize B and Tcells of the donor. Individual donor leukocytes and the antibodiesand/or labels bound to them can be assessed by flow cytometry aftercontacting the donor leukocytes with the recipient antibodies.

The assessment methods are suitable for use with recipients which arevertebrates, preferably being a human.

Non-specific binding between recipient antibodies and Fc receptors ondonor leukocytes can inhibited using known reagents. The donorleukocytes are preferably alive at the time they are contacted with theantibodies obtained from the recipient.

BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a table illustrating results of experimental results describedin Example 5 herein.

FIG. 2 is a table illustrating results of experimental results describedin Example 6 herein.

DETAILED DESCRIPTION

The disclosure relates to methods for crossmatching tissue from a donorwith a suitable recipient for the tissue. The methods involvedetermining anti-donor IgG subtype(s) present in a sample that containsantibodies obtained from the recipient, such as serum prepared from theblood of the recipient.

The donor tissue can be tissue that has already been collected (andpotentially stored and/or frozen) from a living or recently deceasedvertebrate, such as a human donor, or it can be tissue that remains apart of a donor candidate. In any of these circumstances, the individualfrom whom, or from whose corpse, the tissue has been collected or may inthe future be collected is referred to herein as the “donor.” Thecrossmatching method will normally be performed after the donor tissuehas been collected from the donor; however, the method can be performedprior to collecting the tissue to be transplanted (e.g., if that tissuewill not be collected if the recipient is not a suitable recipient forit) by testing leukocytes obtained from the donor against IgG antibodysubtypes in serum obtained from a candidate recipient (i.e., andforegoing harvesting of the tissue if anti-donor IgG1 or IgG3 antibodysubtypes are detected in the candidate recipient's serum). Potentialdonor leukocytes can be frozen and archived, optionally together with orwithin a stored donor tissue.

The method involves contacting leukocytes (e.g., peripheral bloodmononuclear cells) obtained from the donor with antibodies in a sample(e.g., blood serum) obtained from the a potential recipient of thedonor's tissue and there after detecting—in a single assay—whetherantibodies (Abs) of multiple IgG subtypes present in the recipient'ssample have bound to the donor's leukocytes. This assessment ispreferably performed by flow cytometry using reagent that contains aplurality of differentially-labeled Abs, each of which bindsspecifically to only certain subtypes (preferably only to a singlesubtype) of IgG antibodies. Such differentially-labeled Abs aredescribed herein as “distinguishably recognizing” the various subtypesof IgG antibodies. After excess reagent is separated from theleukocytes, the leukocytes are assessed, by flow cytometry for example,to detect any label(s) associated with the leukocytes. From thisinformation, it can be determined which donor-leukocyte-binding IgGsubtypes are present in the recipient's serum. If desired, the variousdonor leukocytes can be distinguished from one another, such as by knownflow cytometric methods, by labeling them with one or more Abs whichdistinguishably recognize (i.e., can be used to distinguish) T and Bcells. In the assay described herein, binding of recipient IgG1 and/orIgG3 antibodies with donor T cells generally indicates a less favorablematch than does the same binding with donor B cells, although both typesof binding indicate a less favorable match than does the absence of bothtypes of binding.

Recipient antibodies can be obtained from the recipient's blood, such asby isolating blood serum (i.e., the cell-free fluid component of blood)therefrom. Recipient antibodies can also, or alternatively, be used inor isolated from any other body fluid or wash/lavage fluid known orbelieved to contain antibodies that are representative of the patient'sbody. By way of example, recipient antibodies can be obtained from apleural aspirate, a bronchial lavage, or a peritoneal fluid sample.Regardless of how the recipient antibody-containing sample is obtainedor prepared, it is important that IgG antibodies (at least of the IgG1and/or IgG3 subtypes) that are present in the sample be treated and/orstored in ways which preserve the ability of IgG1 and or IgG3 antibodiestherein to bind with the donor's leukocytes. A preferred sample ofrecipient antibodies is blood serum.

If the recipient's sample contains donor-leukocyte-binding IgG Abs ofthe IgG1 or IgG3 subtypes, the potential recipient is likely not asuitable recipient for the donor's tissue, and the donor's tissue shouldnot be grafted with the donor. Occurrence of anti-donor IgG1 or IgG3subtypes in the recipient's sample is an indication that graft-rejectionsymptoms are likely to occur if a tissue from the donor is transplantedto the recipient. That is, such occurrence indicates thatantibody-mediated rejection is more likely to occur than if therecipient's sample does not contain anti-donor IgG1 or IgG3 subtypes.The likelihood of graft-rejection symptoms can also be at least roughlycorrelated with increasing degree of detection of IgG1 and/or IgG3.Thus, for example, an individual whose leukocytes provoke relatively lowlevels of binding of recipient IgG1 and/or IgG3 antibodies in the assaydescribed herein would be considered a preferable tissue donor thananother individual whose leukocytes provoke a substantially higher levelof binding of recipient IgG1 and/or IgG3 antibodies in that assay. Also,given the known greater propensity of IgG3 to induce complementfixation, detection of a given level of recipient-IgG3 binding withdonor leukocytes (e.g., two-fold above normal background level) isconsidered an indication of a less favorable donor match than detectionof the same level of recipient-IgG1 binding with the donor's leukocytes.In general, detection of recipient-IgG3 or -IgG1 binding with donorleukocytes greater than about two-fold greater than the basal (i.e.,normal background) level of such binding is considered indicative of anunfavorable donor match (i.e., increased likelihood of graft-rejectionsymptoms), with greater detected binding generally indicating amore-unfavorable donor match.

If the recipient's sample does not contain donor-leukocyte-binding IgGAbs of the IgG1 or IgG3 subtypes, the potential recipient likely is asuitable recipient for the donor's tissue, and the donor's tissue can begrafted to the recipient with a reasonable expectation that it will notbe rejected, at least by physiological processes involving complementfixation.

The methods described herein are therefore useful for determiningwhether an individual recipient can be expected to reject a tissueobtained from a different individual donor (of a different or,preferably, the same species). A recipient can be expected to reject atissue obtained from a donor if the recipient's sample includesdonor-leukocyte-binding antibodies of subtypes IgG1 or IgG3, and can beexpected not to reject the tissue if the donor's sample does not includedonor-leukocyte-binding antibodies of subtypes IgG1 or IgG3 (even if thedonor's sample includes donor-leukocyte-binding antibodies of subtypesIgG2 and/or IgG4).

In contrast to prior methods, the methods described identify not onlylikely-successful tissue grafts which could be identified using priormethods that relied on detection of donor-leukocyte-binding antibodiesof IgG of undifferentiated subtypes, but also additionallikely-successful tissue grafts which could not be identified usingthose prior methods.

The present methods have the advantage that multiple assessments (e.g.,detection of IgG1 and detection of IgG3) can be performed in a singlereaction mixture, using a single aliquot of sample. Because all of themultiple assessments are performed using the same sample, difficultieswhich can arise from reliance of multiple assays on multiplecorresponding controls are avoided.

The present methods have the additional advantage that they areperformed using whole donor cells, rather than synthetic beads to whichdonor antigens are attached. Donor cells (e.g., live PBMCs) can beexpected to closely mimic antigen presentation by donor tissue; bycontrast, synthetic beads bearing donor antigens may present donorantigens substantially differently than the manner in which those sameantigens are presented by donor tissue. Thus, the methods describedherein can be expected to be more accurately predictive of recipientresponses to donor tissue.

Use of the methods described herein for matching human recipients withhuman donors of tissues is intended. However, it is also understood thatthe same methods, with merely routine modifications, can be used tomatch individual recipients of substantially any vertebrate species withdonors of tissues of the same species. Likewise, the same methods, withroutine modifications, can be used to match individual recipients of afirst vertebrate species with tissues obtained from donors of a second,different vertebrate species (although it is recognized that otherassays for recipient appropriateness would likely be conducted forinter-species tissue transfers).

EXAMPLES

The subject matter of this disclosure is now described with reference tothe following Examples. These Examples are provided for the purpose ofillustration only, and the subject matter is not limited to theseExamples, but rather encompasses all variations which are evident as aresult of the teaching provided herein.

Example 1

Isolation of PBMCs from Donor Blood

Peripheral Blood Mononuclear Cells (PBMCs, which include bothlymphocytes and monocytes) were isolated from donor whole blood that wascollected in acid citrate dextrose (ACD) vacutainer tubes. Donor bloodwas transferred to 15 milliliter conical tubes and mixed with 2milliliters methyl cellulose (1% solution, weight/volume; Sigma-Aldrich,St. Louis, Mo., USA) by inversion. The tube was rotated in a 37 degreesCelsius incubator for 15 minutes and left standing at 37 degrees Celsiusfor an additional 30 minutes to separate the plasma layer containingenriched lymphocytes from the red blood cells layer. The plasma layerwas carefully transferred to a fresh 15 milliliter conical tube anddiluted with dulbecco's phosphate-buffered saline (DPBS, Lonza,Walkersville, Md., USA) at a ratio of 1:1 (plasma:DPBS) and mixed byinversion.

Using a Pasteur pipette, the diluted plasma was underlaid withlymphocyte separation medium (LSM) such that the ratio of dilutedplasma:LSM was 1:1. The tube was centrifuged at 22 degrees Celsius in aswing bucket rotor at 400.times.g for 25 minutes with slow deceleration.The clear white band at the interface containing PBMCs was transferredto a fresh 15 milliliter conical tube and diluted with DPBS to a finalvolume of 10 milliters. The tube was centrifuged at 260.times.g for 10minutes at 22 degrees Celsius to pellet the PBMCs. The supernatantcontaining platelets was discarded, leaving the pellet rich in PBMCs.The pellet was carefully resuspended in 2 milliliters DPBS, and thecells were counted in a hemocytometer using 1:2 dilution of Trypan Blue.

IgG Subtype Assay

IgG subtype analysis was carried out using the highly sensitiveflow-cytometry crossmatch (FCXM) platform, as follows. Briefly, PBMCsisolated from the donor blood were aliquoted into separate 5 milliliterpolystyrene round bottom tubes at a concentration of 5.times.10.sup.5cells/tube. The cells were washed with DPBS and pelleted bycentrifugation at 800.times.g for 5 minutes at 22 degrees Celsius. TheFC receptors on the cell surface were blocked using Fc Receptor Blockerreagent (obtained from Innovex Biosciences, Richmond, Calif., USA) for10 minutes at room temperature (ca. 20 degrees Celsius). The blockingreagent was removed by washing the cells twice with DPBS followed byincubation with patient (i.e., recipient) serum for 30 minutes at 4degrees Celsius. Donor cells were also incubated with negative andpositive control serum samples. Positive control sera were prepared bypooling sera from five patients who were highly sensitized (each havingcPRA values greater than 98%). This positive sera reacts with PBMC cellsfrom all donors yielding consistently positive FCXM results. Negativecontrol sera (obtained from Gemini Bio-Products, West Sacramento,Calif., USA) was collected from healthy male donors of the AB serotypeat FDA-licensed facilities in the United States. This material isdefibrinated from source plasma AB. All donor units are tested for viralmarkers and found to be non-reactive. This sera yields consistentlynegative FCXM results.

Following this incubation, cells were washed with a wash buffercontaining DPBS and 1% fetal bovine serum (FBS, Gemini Bio-products,West Sacramento, Calif., USA). The cells were resuspended in wash bufferand transferred to separate tubes containing a lyophilized customcocktail of differentially-labeled antibodies that specificallyrecognize the various IgG subclasses (obtained from BD Lyotube, BDPharmingen, San Jose, Calif., USA). Incubation of donor cells with thelyophilized antibody cocktail was carried out in the dark for 20minutes, and the cells were then washed with wash buffer to remove theexcess antibody.

The specific anti-HLA IgG subtype antibodies bound to the cells weredetected using a multi-color flow cytometry detection method describedherein. IgG subtype levels in patient serum was compared to the levelsobserved in the Negative control serum. The fold change in the IgGsubtype levels in the patient serum over Negative control serum wascalculated. Levels of IgG subtypes in the Patient sera that were 2-foldabove the levels in the Negative control serum were designated aspositive for that subtype.

Example 2 Protocol for Identification of IgG Subtypes by Flow Cytometry

The following description is similar to the procedures described inExample 1, with some minor variations.

Isolate PBMC from whole blood or using frozen PBMC.

Wash twice with phosphate-buffered saline (PBS, pH 7.4) and perform aviability count.

Centrifuge at 1500 rpm for 5 minutes.

Discard the supernatant and resuspend the cell pellet in 0.3 ml FcRblocker reagent per 10.sup.7 cells (Innovex Cat# NB309).

Incubate at room temperature (ca. 20 degrees Celsius) for 10 minutes.

Wash with excess PBS twice.

Resuspend the cells in 10.sup.7 cells/ml in staining buffer (i.e.,phosphate-buffered saline, pH 7.4, containing 5% v/v fetal bovineserum).

Aliquot 100 microliters per test (10.sup.6 per test) of cell suspensionto each 12.times.75 mm tube.

Add 20 microliters neat serum to each tube.

Incubate at 4 degrees Celsius for 30 minutes.

Wash with 2.5 ml staining buffer.

Discard the supernatant.

Resuspend in 100 microliters staining buffer and transfer the content tothe lyotube.

Incubate at room temperature for 20 minutes.

Wash with 2.5 ml staining buffer.

Discard the supernatant and resuspend in 0.4 ml staining buffer.

Acquire the sample, measuring amounts of individual IgG subtypes.

Example 3 Using IgG Subtyping in Heart Transplantation Across a PositiveFlow-Cytometry Cross Match (FCXM)

A positive FCXM is often a deterrent to heart transplantation due to therisk of hyperacute rejection and antibody-mediated rejection (AMR).While highly sensitive to the presence of donor-specific antibodies,FXCM does not determine whether these antibodies bind complement or not.

Case Report: 61 year old African-American male with coronary arterydisease developed following myocardial infarction and coronary arterybypass graft, who developed heart failure due to ischemic cardiomyopathywith ejection fraction (EF) 15-20%. Due to progressive symptoms despitemedical therapy and implantation of a cardiac resynchronization therapydevice, he had a Heart Mate II left ventricular assist device (LVAD)implanted as a bridge to transplant. Post-LVAD course was complicated byrecurrent gastrointestinal bleed requiring multiple transfusions as wellas a driveline exit site infection. He was upgraded to status 1A butunable to get a donor due to high panel reactive antibodies (PRAs)—ClassI 69%, Class II 3%. Desensitization was attempted using plasmapheresis,intravenous immunoglobulin, rituximab, mycophenolate and bortezimib withno significant response.

A suitable donor was identified with a negative complement-dependentcytotoxicity result on prospective cross matching. However, FCXM wasstrongly positive for both T- and B-cells (median channel shift: T-cell362/50, B-cell 359/100). Recipient serum analysis was performed usingcustom antibodies that recognize the different IgG subtypes. Of the 4IgG subtypes, only subtypes 2 and 4 were identified in the recipient'sserum. Since only IgG subtypes 1 and 3 are known to becomplement-binding, it was felt safe to proceed with transplant.

Heart transplantation was performed and recipient received inductionimmunosuppressive therapy (“induction therapy”) with basiliximab as wellas protocol immunosuppression with prednisone, tacrolimus andmycophenolate. He had normal graft function immediately post-op with nohyperacute rejection. Repeat echo done 12 days post-transplant showednormal biventricular function. He had 14 protocol endomyocardialbiopsies over 12 months with no significant cellular rejection seen.Staining of all specimens for C4d and CD68 did not show evidence ofantibody-mediated rejection. He continues to do well with preservedgraft function EF 70% at his first annual visit. A regadenoson stresstest performed at that time showed no evidence of ischemia.

Described in this example is a case where IgG subtyping was utilized toproceed with heart transplantation despite a positive FCXM. The absenceof complement-binding IgG subtypes 1 and 3 on recipient serum led to asuccessful heart transplant without occurrence of rejection and normalgraft function at 1 year.

Example 4 Method of Preparing a Reagent Suitable for Simultaneous FlowCytometric Identification of Four IgG Subtypes

1. Purchase individual anti-IgG 1, 2, 3, 4 antibodies (Abs) that arefluorescently labeled from different companies and determine the clonesof these Abs.

2. Assess each clone to be suitable for the experiment (binding of thesecondary anti-IgG Ab to the primary Ab attached to the HLA antigen (Ag)on PBMC).

3. Determine amount of each secondary anti-IgG Ab that needs to beincluded in the reagent. This requires performing the experimentindividually with each anti-IgG Ab.

4. Perform the experiment with combinations of the 4 anti-IgG Abs (ingroups of 2, 3 or all 4 together) and determine if there is any sterichindrance that prevents binding of the secondary Abs to the primary Absthat are bound to the HLA Ag on PBMCs. If so, select one or moredifferent Abs in place of at least one of the sterically hindered Abs.

5. Select a reagent for blocking the Fc receptor as a backup. Thereagent currently used is the “Fc Receptor Blocker” which iscommercially available, as described above, and works as perexpectations. Others are known in the art.

6. Obtain “Standard Beads” that are labeled with individual IgG subtypesfor use as internal experimental standards to be run with eachexperiment.

7. Perform the standardization of the above reagents with Standard Beadsand freshly isolated PBMCs. Standardization of reagents entailsperforming multiple experiments using the same concentration of anti-IgGAbs in combination as determined above with the same amount of StandardBeads but using PBMCs isolated from various donors. This demonstratesthat there is consistency in the results with Standard Beads for eachexperiment performed and that this assay can detect IgG subtype levelsbound to PBMCs isolated from various donors.

8. Validate the above standardized experiment using freshly isolatedPBMCs from multiple donors (e.g., 20 or more donors).

Each of these steps is preferably document so that details are availablefor inspection by regulatory agencies, if necessary or desirable.

Example 5 An IgG Subtype Specific Flow Crossmatch Assay Can Increase theNumber of Successful Transplants in Sensitized Renal Recipients

A positive flow cytometric crossmatch (FXCM) is considered acontraindication to a successful renal transplant. The standard FXCMdoes not distinguish between the various subtypes of the immunoglobulinmolecule (IgG1, IgG2, IgG3, and IgG4). Only IgG1 and IgG3 subtypes arecapable of maximal complement activation. IgG2 and IgG4 are relativelybenign. We present preliminary results from our study evaluating a newFCXM test that specifically detects and quantifies IgG subtypesresponsible for a positive crossmatch.

The methods used in the experiments described in this Example are nowdescribed. Pre-transplant sera from 7 recipients and blood samples fromtheir respective donors were evaluated. IgG subtype analysis was carriedout using the FCXM. PBMCs isolated from the donor samples were incubatedwith the patient and control sera. The cells were then incubated in thelyophilized custom cocktail of antibodies that specifically recognizethe various IgG subtypes bound to the cells, followed by FCXM analysis.Clq (complement activation) testing on all sera was carried out.

The results of these experiments were as follows. Standard FCXM werepositive in most of the cases studied (5/7), and a transplant wouldtherefore normally not have been conducted based on those results alone.However, using the IgG subtyping assay described herein, we were able todetermine that the positive crossmatch result was attributable to thepresence of non-complement binding IgG2 or IgG4 antibodies and that thetransplant could be performed. There was almost complete agreementbetween the IgG subtyping and Clq results. All cases showed the presenceof non-complement activating antibodies as responsible for the positiveFCXM (except CF). CFL showed the presence of IgG3 antibodies with anegative Clq; probably the result of denatured antibodies. There were noepisodes of clinical rejection observed in any of the cases orrequirement for dialysis in the first week for any of the recipients.These results are summarized in the table illustrated in FIG. 1. In FIG.1, “Cr.” refers to serum creatine determination.

The experiments described in this Example demonstrate that we havedeveloped an IgG subtype FCXM assay that has the ability to identifypresence of complement activating antibodies. This assay has shownitself to be highly accurate in detecting the IgG subtype(s) causing apositive flow crossmatch. The use of this assay could potentially resultin successful transplants even in the presence of a positive FCXM forhighly sensitized recipients.

Example 6 Increasing Successful Transplants in Sensitized Heart andRenal Recipients Using a New Flow Crossmatch Assay to DistinguishBetween IgG Subtype(s)

Transplant programs are reluctant to carry out transplantation in thepresence of a positive flow crossmatch. The standard Flow CytometricCrossmatch (FCXM) does not discriminate between the various subtypes ofthe immunoglobulin molecule (IgG1, IgG2, IgG3, and IgG4). We havedeveloped a new FCXM assay that is able to specifically detect andquantify the amount of complement-activating (IgG1 and IgG3) andnon-complement activating (IgG2 and IgG4) IgG antibodies. We presentpreliminary results using this assay in two separate models for heartand kidney transplantation. We demonstrate that successfultransplantation in the presence of a positive crossmatch can beaccomplished using this assay that has the ability to distinguishbetween the various IgG subtypes.

The methods used in the experiments described in this Example are nowdescribed. Pre-transplant sera from 7 heart recipients (including theone described in Example 3) and 7 kidney recipients and blood samplesfrom their respective donors were used for this study. IgG subtypeanalysis was carried out using the FCXM. PBMCs isolated from the donorsamples were incubated with the patient and control sera. The cells werethen incubated in the lyophilized custom cocktail of antibodies thatspecifically recognize the various IgG subtypes bound to the cells,followed by FCXM analysis. Clq testing was carried out on all sera.

Results obtained for the kidney recipient patients are described inExample 5 and FIG. 1.

Results obtained for the heart recipient patients were as follows. Amajority of the heart transplant cases studied (6/7) had a positivecrossmatch, and a transplant would therefore normally not not have beenconducted. However, using the IgG subtyping assay described herein, wewere able to determine that the positive crossmatch result wasattributable to the presence of non-complement binding IgG2 or IgG4antibodies and that the transplant could be performed. Clq results werein agreement with crossmatch results in most of these cases. Two cases(JO, MP) were positive for Clq; probably due to prozone effect ofHLA-specific IgM antibodies. All cases had positive 30-day and 90-daysurvival post-transplant with no primary graft dysfunction or >2Rrejection (using the terminology of the International Society of Heartand Lung Transplantation Guidelines for the Care of Heart TransplantRecipients). Two cases (JF, RP) who had documented antibody-mediatedrejection and were treated with induction therapy continued to havenormal graft function. These results are summarized in the tableillustrated in FIG. 2.

The experiments described in this Example and in Example 5 demonstratethat the IgG subtype assay described herein is highly accurate fordetecting the IgG subtype(s) causing a positive flow crossmatch. Theseresults demonstrate for the first time that the assay can facilitatesafe transplants even in the presence of a normally contraindicatingstandard positive flow cross-match. Clinical implementation of our IgGsubtypes assay can have a great impact on increasing the number ofsuccessful transplants carried out, especially in sensitized recipients.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this subject matter has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations can bedevised by others skilled in the art without departing from the truespirit and scope of the subject matter described herein. The appendedclaims include all such embodiments and equivalent variations.

What is claimed is:
 1. A method of grafting a tissue obtained from a vertebrate donor to a recipient, the method comprising contacting leukocytes obtained from the donor with antibodies obtained from the recipient, assessing binding between the donor leukocytes and recipient antibodies of at least IgG subtypes IgG1 and IgG3, and grafting the donor tissue to the recipient if substantially no binding between the donor leukocytes and recipient antibodies of either of IgG subtypes IgG1 and IgG3 is detected.
 2. The method of claim 1, comprising assessing binding between the donor leukocytes and recipient antibodies of at least IgG subtypes IgG1, IgG2, IgG3, and IgG4.
 3. The method of claim 2, wherein assessment of binding between the donor leukocytes and recipient antibodies of each of IgG subtypes IgG1, IgG2, IgG3, and IgG4 is performed in a single assay.
 4. The method of claim 1, wherein the assessment of binding between donor leukocytes and the antibodies is performed by contacting recipient antibodies with differentially-labeled antibodies that specifically recognize antibodies of the IgG1 and IgG3 subclasses.
 5. The method of claim 1, wherein the assessment of binding between donor leukocytes and the antibodies is performed by contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses.
 6. The method of claim 5, further comprising contacting the donor leukocytes with differentially-labeled antibodies that distinguishably recognize B and T cells of the donor.
 7. The method of claim 1, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies.
 8. The method of claim 1, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies and after contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses.
 9. The method of claim 1, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies, after contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses, and after contacting the donor leukocytes with differentially-labeled antibodies that distinguishably recognize B and T cells of the donor.
 10. The method of claim 1, wherein the recipient is a vertebrate.
 11. The method of claim 10, wherein the recipient is the same species as the donor.
 12. The method of claim 11, wherein the recipient is a human.
 13. The method of claim 1, wherein non-specific binding between recipient antibodies and Fc receptors on donor leukocytes is inhibited.
 14. The method of claim 13, wherein the non-specific binding is inhibited by contacting the donor leukocytes with a peptide that inhibits binding between antibodies and surface Fc receptors prior to contacting the donor leukocytes with the recipient antibodies.
 15. The method of claim 1, wherein the donor leukocytes are alive at the time they are contacted with the recipient antibodies.
 16. A method of assessing compatibility of a body tissue of a potential donor for grafting with a recipient, the method comprising contacting leukocytes obtained from the potential donor with antibodies obtained from the recipient, assessing binding between the potential donor leukocytes and recipient antibodies of at least IgG subtypes IgG1 and IgG3, whereby the body tissue is assessed as compatible for grafting with the recipient if substantially no binding between the donor leukocytes and recipient antibodies of either of IgG subtypes IgG1 and IgG3 is detected.
 17. The method of claim 16, comprising assessing binding between the donor leukocytes and recipient antibodies of at least IgG subtypes IgG1, IgG2, IgG3, and IgG4.
 18. The method of claim 17, wherein assessment of binding between the donor leukocytes and recipient antibodies of each of IgG subtypes IgG1, IgG2, IgG3, and IgG4 is performed in a single assay.
 19. The method of claim 16, wherein the assessment of binding between donor leukocytes and the antibodies is performed by contacting recipient antibodies with differentially-labeled antibodies that specifically recognize antibodies of the IgG1 and IgG3 subclasses.
 20. The method of claim 16, wherein the assessment of binding between donor leukocytes and the antibodies is performed by contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses.
 21. The method of claim 20, further comprising contacting the donor leukocytes with differentially-labeled antibodies that distinguishably recognize B and T cells of the donor.
 22. The method of claim 16, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient antibodies.
 23. The method of claim 16, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient serum and after contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses.
 24. The method of claim 16, wherein individual donor leukocytes are assessed by flow cytometry after contacting the donor leukocytes with the recipient serum, after contacting recipient antibodies with differentially-labeled antibodies that distinguishably recognize antibodies of the IgG1, IgG2, IgG3, and IgG4 subclasses, and after contacting the donor leukocytes with differentially-labeled antibodies that distinguishably recognize B and T cells of the donor.
 25. The method of claim 16, wherein the recipient is a vertebrate.
 26. The method of claim 25, wherein the recipient is the same species as the donor.
 27. The method of claim 26, wherein the recipient is a human.
 28. The method of claim 16, wherein non-specific binding between recipient antibodies and Fc receptors on donor leukocytes is inhibited.
 29. The method of claim 28, wherein the non-specific binding is inhibited by contacting the donor leukocytes with a peptide that inhibits binding between antibodies and surface Fc receptors prior to contacting the donor leukocytes with the antibodies obtained from the recipient.
 30. The method of claim 1, wherein the donor leukocytes are alive at the time they are contacted with the antibodies obtained from the recipient. 